The Department of Biological Sciences seeks a NIH-NCRR Shared Instrumentation grant to purchase a flow cytometer/cell sorter for its Flow Cytometry Facility. The instrument requested, a Coulter EPICS Elite ESP, will replace the Facility's current 11-year-old Coulter EPICS 753 dual-laser cell sorter. A new instrument is needed because Coulter is no longer providing parts or service contracts for the 753. In addition, the 753 has become very outdated; many applications require the enhanced capabilities of the Elite ESP. The Elite ESP also is much easier to set up and operate, which will allow trained users to independently operate the instrument, expanding the time it will be usable. The Facility will be run by the Flow Cytometry Facility Committee, consisting of the principle investigator, co-P.I., two other major users, and the Facility operator. The Biological Sciences Flow Cytometry Facility has facilitated NIH-supported investigations of a wide variety of basic biomedical research problems in molecular, cell, and developmental biology, genetics, biochemistry, and immunology, studied with cells from a wide range of organisms including mammals, Drosophila, yeast, and bacteria. The six major users and three minor user included in the proposal are all from the School of Humanities and Sciences (one from the Department of Chemistry and the remainder from the Department of Biological Sciences). The projects that will use the new instrument are highly diverse and in some cases represent novel applications of flow cytometry. The P.I., an immunologist interested in genetic variation in immune responsiveness, will investigate mechanisms regulating production of cytokines and other immune effector functions by T cells and macrophages, using immunofluorescence and assays of intracellular signalling molecules. The second major project is focused on mechanisms of DNA damage and repair; investigating the role of p53 in cellular sensitivity to DNA damage, using DNA vital dyes, and assessing nucleotide excision repair in human cells, using shuttle vectors containing Green Fluorescent Protein (GFP) reporter genes. The third project seeks to identify genes involved in protein folding and degradation in animal cells, with the cystic fibrosis transmembrane conductance regulator (CFTR) as the model protein, utilizing fluorescent antibody-detectable FLAG-CFTR constructs. The fourth project will use GFP reporter constructs to identify developmentally-controlled promoters in Rhizobium bacteria solely expressed in symbiotic root nodules, perhaps induced by host signal molecules. The fifth project will use flow cytometry to investigate the chemical reactions involved in the interactions between antigenic peptide, MHC proteins, and T cell receptors, using fluorescence-tagged peptides and sorted T cell populations. The sixth major project will identify proteins involved in the degradative regulation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, using GFP-HMG-CoA reductase fusion proteins to isolate mutant animal cells. The three minor users will utilize flow cytometry in their studies of the genetic control of sexual behavior in Drosophila, the development of the mammalian cerebral cortex, and microtubule function in yeast. This new instrument should contribute greatly to progress in diverse areas of biomedical research.